1. Field of the Invention
The present invention relates to an integral multi-layer analytical element for use in the measurement of alkaline phosphatase activity in a liquid sample, and more particularly to an integral multilayer analytical element for use in the measurement of alkaline phosphatase activity in aqueous liquid samples, particularly in body fluids in the course of clinical tests.
2. Description of Prior Arts
It is very important to measure alkaline phosphatase activity in human body fluids in clinical tests. The measurement of the activity of alkaline phosphatase (hereinafter, sometimes referred to as ALP) in body fluids is made in the diagnoses of bone diseases such as osteoma and hepatopaphy such as obstructive jaundice.
A method for measuring alkaline phosphatase activity was reported by H. D. Kay in J. Biol. Chem. 89 235 (1930). Since then, various methods have been proposed. Among them, a method reported by E. J. King in Biochem, J. 33 1185 (1939) has given a remarkable effect of simplifying the measurement, in which aryl phosphates such as self-developable p-nitrophenyl phosphate are used as substrates.
The above measuring methods have been improved by research workers. It has been found that the optimum pH of color developing reaction is about 10, that there is a preferred type of alcohol to be allowed to coexist, and that an inorganic salt, particularly magnesium ion is preferably used as activator. The improved method is established as IFCC method [Clin. Chimica Acta (1983) 339F-367F] wherein disodium p-nitrophenyl phosphate is used as substrate.
However, the aforementioned method has disadvantages in that a solution of disodium p-nitrophenyl phosphate must be prepared at the time of measurement and is unstable and that the preservable period of the solution at 20.degree. to 26.degree. C. is only 8 hours at most. Further, disodium p-nitrophenyl phosphate is unstable even at a freezing temperature as reported by Amador E., et al. [J. Amer. Med., Ass. 184 953 (1963)]. Therefore, it is necessary to assay the purity of reagent just before the measurement and the reagent must be carefully preserved during the time from its preparation to the measurement.
The aforementioned problem of instability associated with p-nitrophenyl phosphate has been greatly improved by the use of organic amine salts of p-nitrophenyl phosphate disclosed in Japanese Patent Publication No. 45(1970)34872. Further, amine salts of thimolphthalein monophosphate, which have absorption spectrum in the wavelength region longer than that of p-nitrophenyl phosphate, have been synthesized by John M. Elickson et al. (Clinical Chemistry Vol. 19, No. 6, 1973, p-664). Preservability of the substrates per se has been greatly improved by these studies, and it has become possible to easily incorporate the substrate into a kit for ALP measurement.
An analytical method which can been rapidly carried out with a simple operation, has been highly desired by medical persons using results of clinical tests. For this purpose, dry analytical methods have been used in recent years, and particularly there have been developed and improved ALP measuring methods using an integral multilayer analytical element which is easy to handle.
As a typical example of the element, there is known an integral multilayer analytical element wherein a water-absorbing reagent layer containing a color reaction reagent and a hydrophilic polymer binder is provided on a transparent support and a porous spreading layer as the uppermost layer is provided on said reagent layer, proposed by B. Walter [Analytical Chemistry (1983) 55 493A]. Further, a number of elements are known.
In performing the analysis of ALP by dry analytical methods, however, the substrates such as p-nitrophenyl phosphate in the aforementioned wet analytical methods are not considered to be fully satisfactory with respect to an improvement in preservability. Accordingly, it is highly demanded to develop an integral multilayer analytical element for the measurement of alkaline phosphatase activity, in which the preservability of the substrate is satisfactorily improved.